Application of SFRP1 gene in buffalo ovarian granular cells

Abstract

The invention discloses application of an SFRP1 gene in buffalo ovarian granular cells, and belongs to the technical field of cell engineering and gene engineering. According to the invention, SFRP1 is taken as a research object, an SFRP1 overexpression vector or siRNA-SFRP1 is respectively transfected to ovarian granular cells by constructing the SFRP1 overexpression vector and synthesizing small interfering RNA (siRNA-SFRP1), then changes of signal channel gene mRNA and protein level related to ovarian granular cell apoptosis, proliferation and E2 and P4 secretion are detected, and finally phenotypic changes of the ovarian granular cells are detected. Results show that the SFRP1 can inhibit apoptosis of ovarian granular cells and promote proliferation and E2 and P4 secretion. The application of the SFRP1 in the ovarian granular cells is explored, and the SFRP1 has good application in research of an ovarian follicle atresia mechanism, initial estrus initiation and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of cell engineering and gene engineering, and relates to the application of SFRP1 gene in buffalo ovarian granulosa cells. Background Art

[0002] Granulosa cells are an important component of follicles and the main site of estrogen synthesis. During follicular development, they gradually proliferate from a single flat layer to a multi-layer columnar layer, and produce cytokines, growth factors, and estrogens related to follicular development, promoting the maturation of follicles. It is currently believed that the recruitment, selection, and ovulation of follicles are essentially the proliferation and differentiation of granulosa cells, while the apoptosis of granulosa cells represents the atresia and degeneration of follicles.

[0003] Secreted frizzled-related protein 1 (SFRP1), also known as frizzled-related protein (FRP), FRP1, FrzA, secreted apoptosis-related protein 2 (SARP2), was first discovered by Melkonyan et al. in mice in 1997. This protein belongs to the secreted fizzled-related protein family (SFRPs). The SFRPs family mainly includes SFRP1, SFRP2, SFRP3 (also known as FrzB), SFRP4 and SFRP5, which are expressed in different cells and organs. SFRPs is also a family of proteins that can block the Wnt signaling pathway. The SFRP1 gene is widely expressed in the human body, and the expression levels are from high to low in the endometrium, fat, salivary glands, kidneys, heart, gallbladder, prostate, bladder, ovaries, testes, bone marrow, skin, and pancreas. SFRP1 can be folded into two domains: the C-terminal carboxyl-terminal netrin-like region (NTR-like) and the N-terminal amino-terminal cysteine-rich region (frizzled-like cysteine-rich domain, z-CRD). Among them, CRD has certain similarities with the binding site of frizzled protein (Fz) receptor in the Wnt signaling pathway, competing with Wnt ligands to bind to Fz receptors, hindering Wnt signal transduction, and the Wnt signaling pathway plays an important role in the embryonic development of multicellular organisms and cell proliferation, differentiation and apoptosis in adult tissues. SFRP1 negatively regulates the Wnt pathway and participates in regulating complex processes such as cell proliferation, differentiation, polarization, apoptosis, migration, carcinogenesis, growth and development of the body, and diseases. It is abnormally expressed in many diseases, such as ovarian cancer, periodontitis, oral submucous fibrosis, glioma, acute lymphocytic leukemia, cervical cancer, osteosarcoma, gastric cancer, liver cancer, breast cancer, etc. Summary of the invention

[0004] The purpose of the present invention is to provide an application of SFRP1 gene in buffalo ovarian granulosa cells in response to the above problems. The present invention takes SFRP1 as the research object, constructs an SFRP1 overexpression vector and a synthetic small interfering RNA (siRNA-SFRP1), and transfects the SFRP1 overexpression vector or siRNA-SFRP1 into ovarian granulosa cells, respectively, and then detects the changes in the mRNA and protein levels of signaling pathway genes related to ovarian granulosa cell apoptosis, proliferation, and E2 and P4 secretion, and finally detects the phenotypic changes of ovarian granulosa cells. The results show that SFRP1 can inhibit the apoptosis of ovarian granulosa cells and promote proliferation and E2 and P4 secretion. By exploring the application of SFRP1 in ovarian granulosa cells, the present invention has a good application in studying the mechanism of ovarian follicle atresia and the initiation of puberty.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] One object of the present invention is to provide the application of SFRP1 gene in buffalo ovarian granulosa cells, and the application of SFRP1 gene in ovarian granulosa cell proliferation, apoptosis, E2 secretion and / or progesterone secretion in vitro.

[0007] To further explain, the application is:

[0008] In vitro, increasing the SFRP1 gene can promote the proliferation of ovarian granulosa cells, and / or decreasing the SFRP1 gene can inhibit the proliferation of ovarian granulosa cells;

[0009] In vitro, increasing SFRP1 gene can inhibit apoptosis of ovarian granulosa cells, and / or decreasing SFRP1 gene can promote apoptosis of ovarian granulosa cells;

[0010] In vitro, increasing the SFRP1 gene can inhibit E2 secretion in ovarian granulosa cells, and / or decreasing the SFRP1 gene can promote E2 secretion in ovarian granulosa cells;

[0011] In vitro, increasing the SFRP1 gene can promote the secretion of progesterone by ovarian granulosa cells, and / or decreasing the SFRP1 gene can inhibit the secretion of progesterone by ovarian granulosa cells; the progesterone is progesterone.

[0012] The present invention also provides another purpose of using the SFRP1 gene in the preparation of a drug for promoting the secretion of progesterone by ovarian granulosa cells, specifically by increasing exogenous SFRP1 gene.

[0013] It is further explained that the increase of exogenous SFRP1 gene is achieved by the following method: connecting the SFRP1 gene to a vector to construct an overexpression vector containing the SFRP1 gene; and then transfecting the overexpression vector containing the SFRP1 gene into ovarian granulosa cells.

[0014] The present invention also provides a third purpose of using the SFRP1 gene in buffalo ovarian granulosa cells. In vitro, adding the SFRP1 gene can improve the maturation rate, cleavage rate, blastocyst rate and blastocyst quality of buffalo ovarian granulosa cells.

[0015] It is further explained that the increase of SFRP1 in the application of the present invention is achieved by the following method: constructing an overexpression vector containing the SFRP1 gene, and transfecting the overexpression vector into buffalo ovarian granulosa cells; the reduction of SFRP1 is achieved by the following method: transfecting buffalo ovarian granulosa cells with siRNA that inhibits the expression of SFRP1 gene.

[0016] The fourth object of the present invention is to provide an application of siRNA for inhibiting SFRP1 gene in buffalo ovarian granulosa cells, wherein the application environment is an in vitro environment; siRNA can inhibit apoptosis of ovarian granulosa cells, promote proliferation of ovarian granulosa cells, inhibit E2 secretion in ovarian granulosa cells, and promote progesterone secretion in ovarian granulosa cells;

[0017] The sequence of siRNA that inhibits SFRP1 gene is as follows:

[0018] siRNA-SFRP1-2:

[0019] siSFRP1-2-sense(5'-3'):GAGAUGCUCAAGUGCGACA,

[0020] siSFRP1-2-antisense(5'-3'):UGUCGCACUUGAGCAUCUC.

[0021] Due to the adoption of the above technical solution, the present invention has the following beneficial effects:

[0022] The results obtained by the inventors through research are: overexpression of SFRP1 in buffalo granulosa cells will reduce the expression of genes such as CTNNB1, Gsk3B, Caspase3, Bcl2, Cyp19a1, C-myc, CCND2, DVL1, WNT2, WNT10, SFRP2, and promote the expression of Axin2 and Bax genes. Overexpression of SFRP1 in buffalo granulosa cells will promote p-GSK3β and inhibit the expression of proteins such as β-catenin, p-AKT, and LEF1. Overexpression of SFRP1 in buffalo granulosa cells will inhibit granulosa cell proliferation, reduce the expression of E2, and promote the expression of progesterone. After interfering with the SFRP1 gene, the opposite experimental results were obtained. The oocytes were matured in the granulosa cells of the SFRP1 overexpression group, the SFRP1 interference group, and the control group. The SFRP1 interference group significantly improved the maturation rate, cleavage rate, blastocyst rate, and blastocyst quality (OCT4 expression is positively correlated with embryo quality), and the SFRP1 overexpression group had the opposite effect. It shows that by interfering with the SFRP1 gene, the proliferation of granulosa cells and the expression of E2 can be promoted, thereby inhibiting follicular atresia and increasing the synchronized estrus ovulation rate; on the other hand, interfering with the SFRP1 gene to make a single layer of granulosa cells can promote the maturation rate, cleavage rate, blastocyst rate and blastocyst quality, thereby improving the efficiency of embryo culture in vitro, which has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a comparative diagram of the time screening of overexpression and interference of SFRP1 in buffalo granulosa cells of the present invention. The upper left figure is a diagram of SFRP1 gene overexpression; the upper right figure is a diagram of SFRP1 gene interference; and the lower figure is the red fluorescence expression after transfection of SFRP1.

[0024] Figure 2 This is a diagram showing the gene expression changes of overexpressed SFRP1 in buffalo granulosa cells of the present invention.

[0025] Figure 3 The diagram is a protein expression change diagram of overexpression and interference of SFRP1 in buffalo granulosa cells of the present invention. The left diagram is a WB diagram; the right diagram is a WB relative expression level.

[0026] Figure 4 This is a comparison diagram of the promotion of cell proliferation by overexpression of SFRP1 in buffalo granulosa cells of the present invention.

[0027] Figure 5 This is a comparison diagram of the changes in hormone levels of buffalo granulosa cells overexpressing SFRP1 in the present invention. The left figure is a comparison diagram of the effects on progesterone hormone; the right figure is a comparison diagram of the effects on secretion of E2 hormone.

[0028] Figure 6This is a comparison diagram of the effects of overexpression or interference of SFRP1 in buffalo granulosa cells on IVF embryo culture. The upper diagram is a comparison diagram of the number of mature, cleavage, and blastocysts; the lower diagram is an immunofluorescence diagram of the effects on Oct4 protein expression. DETAILED DESCRIPTION

[0029] The specific implementation of the invention is further described below with reference to the accompanying drawings.

[0030] According to the NCBI database, primers were designed and cloned into pDC316 with eGFP as follows: SFRP1-pDC316-F (5'-3'): acgcagatcgaattcATGGGCGACGGGCGCTGG, SFRP1-pDC316-R (5'-3'): tgctcaccatCTTGAAGACCGACTGGAAGGTG, EGFP-SFRP1-pDC316-F (5'-3'): ggtcttcaagATGGTGAGCAAGGGCGAGG, EGFP-SFRP1-pDC316-R (5'-3'): gacgagctcaagcttTTACTTGTACAGCTCGTCCATGCC. RNA was extracted from buffalo ovaries, reverse transcribed into cDNA, and amplified by PCR. eGFP was amplified using pEGFP-N1 as a template to construct the pDC316-SFRP1-eGFP vector, which was sent for sequencing and the vector with the correct sequence fragment was screened for subsequent experiments.

[0031] pass Figure 1 (Top left figure) It can be seen that the overexpression of SFRP1 in buffalo granulosa cells at 24, 48 and 72 hours was significantly different from the control fragment, and the best time was 24 hours; Figure 1 (Upper right figure) It can be seen that the best interference fragment transfected buffalo granulosa cells at 24, 48 and 72 hours were significantly different from the control fragment, and the best time was 24 hours. Figure 1 (Figure below) It can be seen that red fluorescence is expressed at 24, 48 and 72 hours, and the fluorescence increases with time.

[0032] 2. Design of synthetic interference fragments

[0033] Utilizing ThermoFisher siRNA Design Website ( InvitrogenBlock-iTRNAiDesigner (thermofisher.com) ) Designed three buffalo siRNA fragments, namely

[0034] siSFRP1-1-sense(5'-3'):CAUGUGACAACGAGCUGAA,

[0035] siSFRP1-1-antisense(5'-3'):UUCAGCUCGUUGUCACAUG;

[0036] siSFRP1-2-sense(5'-3'):GAGAUGCUCAAGUGCGACA,

[0037] siSFRP1-2-antisense(5'-3'):UGUCGCACUUGAGCAUCUC;

[0038] siSFRP1-3-sense(5'-3'):AGCUGGUCCUAUACCUGAA,

[0039] siSFRP1-3-antisense(5'-3'):UUCAGGUAUAGGACCAGCU;

[0040] Control group siNC-sense (5′-3′): UUCUCCGAACGUGUCACGU;

[0041] siNC-antisense (5'-3'): ACGUGACACGUUCGGAGAA. Synthesized by Zixi Biotechnology Co., Ltd.

[0042] 3. Isolation and Culture of Buffalo Oocytes and Granulosa Cells

[0043] Buffalo ovaries were obtained from a slaughterhouse and rinsed three times with 2× double-antibody saline. Follicles with a diameter of 2-4 mm were selected, and follicular fluid was extracted with a syringe to collect oocytes for subsequent experiments. Granulosa cells were collected by centrifugation of the remaining follicular fluid and cultured in a serum-free culture system until they adhered to the wall.

[0044] 4. Screening of interference fragments and interference time

[0045] Buffalo granulosa cells were collected at 1×10 4 1000 cells / well were inoculated in a 24-well plate. Transfection was performed according to the instructions of the siRNA transfection reagent LipofectamineRNAiMAX. Cells were collected 24 hours after transfection, and the best interference fragment was screened by detecting the effect of the interference fragment on the expression of the buffalo SFRP1 gene. The best interference fragment and the control fragment were transfected into buffalo granulosa cells, and the cells were collected 24, 48 and 72 hours after transfection to screen the best interference time.

[0046] After screening, the best interference fragment was siRNA-SFRP-2. Figure 1It can be seen that the upper left figure is the overexpression time screening figure, and the optimal time is 24h; the upper right figure shows that the best interference fragment transfected buffalo granulosa cells at 24, 48 and 72h are significantly different from the control fragment, and the optimal time is 48h. The lower figure is the red fluorescence expression of transfection.

[0047] 5. Oocyte Maturation in vitro

[0048] COCs wrapped with 2 or more layers of cumulus cells were selected, and 10 to 20 COCs were placed in a 40 μL in vitro maturation medium (TCM199 + 10% FBS + 5 μg / mL FSH + 10 μg / mL LH + 0.2 mmol / L sodium pyruvate + 1 μg / mL E2 + 50 μmol / L cysteine ​​+ 25 ng / mL EGF) droplet covered with paraffin oil, and inoculated into granulosa cell monolayer cells. The monolayer cells were divided into SFRP1 overexpression group, SFRP1 interference group and control group, and cultured for 22 to 24 hours at 38.5°C, 5% CO2 and saturated humidity. Oocyte maturation is marked by the discharge of the first polar body.

[0049] Maturation rate (%) = number of oocytes with the first polar body released / total number of oocytes incubated × 100%

[0050] The comparison data of cell maturation rate is shown in the table below.

[0051]

[0052] Different lowercase letters on the shoulders indicate significant differences (P<0.05); different uppercase letters on the shoulders indicate extremely significant differences (P<0.01); the same letters on the shoulders indicate no significant differences (P>0.05). The same below.

[0053] 6. IVF embryo production

[0054] Sperm used for IVF was purchased from frozen semen of Mora or Nili-Lafite buffalo produced by the Guangxi Zhuang Autonomous Region Animal and Poultry Breeding Station. After thawing, the sperm was inoculated into the oocytes after removing the cumulus cells. In vitro culture of fertilized eggs and observation of early embryonic development: the fertilized oocytes were transferred into CM solution (TCM199+10% FBS), and the cumulus cells were removed by repeated blowing with a pipette, and then washed once with CM solution, and transferred into granulosa cell monolayer droplets. The granulosa cell monolayer was divided into SFRP1 overexpression group, SFRP1 interference group and control group, and cultured in a CO2 incubator, 5% CO2, 39°C and saturated humidity. The culture medium was replaced by half every 48h, the cleavage rate was recorded 48h after fertilization, and the blastocyst development was recorded 5-9d.

[0055] The comparison data of cell cleavage rate and blastocyst rate are shown in the table below.

[0056]

[0057] The SFRP1 interference group significantly increased the maturation rate, cleavage rate, and blastocyst rate, while the overexpression group did the opposite.

[0058] 7. Immunofluorescence Detection

[0059] Embryos or cells that developed to 7 days old were washed 3 times with PBS, fixed in 4% paraformaldehyde solution at room temperature for 30 minutes, washed 3 times with PBST (PBS containing 0.1% Tween-20), 5 minutes each time, and then transferred to a permeabilization solution containing 0.1% TritonX-100 for 30 minutes, washed with PBST, and then transferred to a blocking solution (PBST+5% BSA) for blocking at room temperature for 1 hour, and then transferred to the primary antibody after washing with PBST. The negative control group was not incubated with the primary antibody and incubated in a humidified box at 4°C overnight; after washing with PBST, the secondary antibody (goat anti-rabbit IgG H&L, 1:1000, goat anti-mouse IgG H&L, 1:1000) was transferred to room temperature for 2 hours, and then washed with PBST and transferred to 10μg / mL Hoechst33342 for nuclear counterstaining for 15 minutes. After washing with PBST, the cells were transferred to a small amount of antifade droplets on a glass slide, covered with a cover slip, fixed, and observed and photographed under a laser confocal microscope. The fluorescence intensity was analyzed using ImageJv1.8.0 software, and the mean fluorescence intensity was used to semi-quantitatively characterize the specific protein expression.

[0060] like Figure 6 As shown, compared with the control group, the si-SFRP1 group promoted the expression of Oct4 protein, while the SFRP1 overexpression group reduced the expression of Oct4 protein.

[0061] 8. Real-time fluorescence quantitative PCR detection

[0062] After collecting the cells of the experimental group and the control group 24 hours after interference, RNA was extracted using an RNA extraction kit, and reverse transcription was performed, and cDNA was diluted to a concentration of 100 ng / μL. Primers were designed using PrimerPremier5.0, as shown in Table 2. Primers were synthesized by Wuhan Qingke Innovation Biotechnology Co., Ltd. Real-time fluorescence quantitative PCR reaction system 20μL: cDNA 100ng, 0.5μL of upstream and downstream primers each, 2×PowerUpSYBR Green MasterMix 10μL, ddH2O supplemented to 20μL. PCR reaction conditions: 95℃ pre-denaturation for 5min; 95℃ denaturation for 10s, 60℃ annealing for 15s, 72℃ extension for 60s, for a total of 40 cycles. Each group of experiments was repeated 3 times, according to 2 -△△Ct The expression level of each gene was calculated relative to the control group, and Graph PadPrism was used for significance analysis and plotting.

[0063] like Figure 2 As shown in the results, overexpression of SFRP1 gene reduced the expression of genes such as CTNNB1, Gsk3B, Caspase3, Bcl2, Cyp19a1, C-myc, CCND2, DVL1, WNT2, WNT10, and SFRP2, and promoted the expression of genes such as Axin2 and Bax.

[0064] 9. CCK8 detection of cell proliferation

[0065] Inoculate the cell suspension in a 96-well plate, about 100 μl per well, and repeat 3 times for the same sample; add 10 μl of CCK-8 solution to each well at 24, 48, and 72 h, mix well, and place the culture plate in an incubator for 1-4 h; measure the absorbance (OD) at 450 nm with an enzyme-labeled instrument.

[0066] like Figure 4 As shown, overexpression of SFRP1 inhibited the proliferation of buffalo granulosa cells, but the difference was not significant.

[0067] 10.E2 and P4 hormone determination

[0068] The collected cell culture fluid, E2 (estrogen) and P4 (progesterone) were determined using ELISA kits. First, the standard curve was drawn, and blank wells, standard wells and sample wells were set. 50 μL of standard diluted with standard diluent (0, 3.75, 7.5, 15, 30, 60, 120 pmol / mL) was added to the standard wells, and 50 μL of cell culture fluid diluted with sample diluent at 1:5 was added to the sample wells. After sealing, the membrane was incubated at 37°C for 30 minutes, the liquid was discarded, and it was washed with PBS 4 to 5 times. After patting dry, 50 μL of enzyme labeling reagent was added to continue sealing the membrane, incubated at 37°C for 30 minutes, and after repeating the washing steps, 50 μL of color developer A and B were added, and the color was developed at 37°C for 15 minutes. Then 50 μL of stop solution was added, and the OD value at 450nm was measured with an enzyme labeler.

[0069] like Figure 5 As shown, overexpression of SFRP1 in buffalo granulosa cells decreased the expression of E2 and promoted the expression of progesterone.

[0070] 11. Western blot detection

[0071] After collecting the cells, centrifuge and discard the supernatant, add RIPA lysis buffer (add 1mmol / LPMSF) to extract the total cell protein, and determine the total protein concentration by BCA method. After the total protein was denatured at 98℃ for 10min, it was loaded for SDS-PAGE electrophoresis, and the gel was transferred to the PVDF membrane by wet transfer method. After blocking for 10min, the PVDF membrane was washed 3 times with TBST. Add the primary antibody (1:1000 dilution), incubate at 4℃ overnight, wash 3 times with TBST, add the secondary antibody (1:1000 dilution), incubate at room temperature for 1h, and wash 3 times with TBST. Prepare the color development solution, add the PVDF membrane and the color development solution to the color development box, react for 3min in the dark, and expose and take pictures in the imager.

[0072] like Figure 3 As shown, overexpression of SFRP1 in buffalo granulosa cells promoted p-GSK3β and inhibited the expression of β-catenin, p-AKT, LEF1 and other proteins.

[0073] 1.4 Statistical analysis

[0074] Each experiment was repeated 3 times, and GraphPad Prism 7.0 was used for significance analysis and drawing. The experimental data were expressed as mean ± standard error, and the two groups of data were compared using t-test. P < 0.05 indicated a significant difference, and P < 0.01 indicated an extremely significant difference.

[0075] 2. Result analysis:

[0076] Overexpression of SFRP1 in buffalo granulosa cells reduces the expression of genes such as CTNNB1, Gsk3B, Caspase3, Bcl2, Cyp19a1, C-myc, CCND2, DVL1, WNT2, WNT10, and SFRP2, and promotes the expression of Axin2 and Bax genes. Overexpression of SFRP1 in buffalo granulosa cells promotes p-GSK3β and inhibits the expression of proteins such as β-catenin, p-AKT, and LEF1. Overexpression of SFRP1 in buffalo granulosa cells inhibits granulosa cell proliferation, reduces the expression of E2, and promotes the expression of progesterone. After interfering with the SFRP1 gene, the opposite experimental results were obtained. The oocytes were matured in the granulosa cells of the SFRP1 overexpression group, the SFRP1 interference group, and the control group. The SFRP1 interference group significantly improved the maturation rate, cleavage rate, blastocyst rate, and blastocyst quality (OCT4 expression was positively correlated with embryo quality), while the SFRP1 overexpression group had the opposite effect. This indicates that interfering with the SFRP1 gene can promote the proliferation of granulosa cells and the expression of E2, thereby inhibiting follicular atresia and increasing the synchronized estrus ovulation rate; on the other hand, interfering with the SFRP1 gene to form a single layer of granulosa cells can promote the maturation rate, cleavage rate, blastocyst rate and blastocyst quality, thereby improving the efficiency of embryo culture in vitro.

[0077] The above description is a detailed description of the preferred feasible embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modified changes completed under the technical spirit suggested by the present invention should fall within the patent scope covered by the present invention.

Claims

1. The application of SFRP1 gene in buffalo ovarian granulosa cells is characterized by: In vitro, the application of SFRP1 gene in ovarian granulosa cell proliferation, apoptosis, E2 secretion and / or progesterone secretion.

2. The use according to claim 1, characterized in that: In vitro, increasing the SFRP1 gene can promote the proliferation of ovarian granulosa cells, and / or decreasing the SFRP1 gene can inhibit the proliferation of ovarian granulosa cells; In vitro, increasing SFRP1 gene can inhibit apoptosis of ovarian granulosa cells, and / or decreasing SFRP1 gene can promote apoptosis of ovarian granulosa cells; In vitro, increasing the SFRP1 gene can inhibit E2 secretion in ovarian granulosa cells, and / or decreasing the SFRP1 gene can promote E2 secretion in ovarian granulosa cells; In vitro, increasing the SFRP1 gene can promote the secretion of progesterone by ovarian granulosa cells, and / or decreasing the SFRP1 gene can inhibit the secretion of progesterone by ovarian granulosa cells; the progesterone is progesterone.

3. Use of the SFRP1 gene in the preparation of a drug for promoting the secretion of progesterone by ovarian granulosa cells, characterized in that: Specifically, it is by adding exogenous SFRP1 gene.

4. The use of the SFRP1 gene according to claim 3 in the preparation of a drug for promoting the secretion of progesterone by ovarian granulosa cells, characterized in that: The increase of exogenous SFRP1 gene is achieved by the following method: connecting the SFRP1 gene to a vector to construct an overexpression vector containing the SFRP1 gene; and then transfecting the overexpression vector containing the SFRP1 gene into ovarian granulosa cells.

5. Application of SFRP1 gene in buffalo ovarian granulosa cells. In vitro, increasing the SFRP1 gene can improve the maturation rate, cleavage rate, blastocyst rate and blastocyst quality of buffalo ovarian granulosa cells.

6. The use according to claim 2 or 5, characterized in that: The increase of SFRP1 is achieved by the following method: constructing an overexpression vector containing the SFRP1 gene and transfecting the overexpression vector into buffalo ovarian granulosa cells; the reduction of SFRP1 is achieved by the following method: transfecting buffalo ovarian granulosa cells with siRNA that inhibits the expression of SFRP1 gene.

7. Use of siRNA for inhibiting SFRP1 gene in buffalo ovarian granulosa cells, characterized in that: The application environment is an in vitro environment; siRNA can inhibit apoptosis of ovarian granulosa cells, promote proliferation of ovarian granulosa cells, inhibit E2 secretion in ovarian granulosa cells, and promote progesterone secretion in ovarian granulosa cells; The sequence of siRNA that inhibits SFRP1 gene is as follows: siRNA-SFRP1-2: siSFRP1-2-sense(5'-3'):GAGAUGCUCAAGUGCGACA, siSFRP1-2-antisense (5'-3'):UGUCGCACUUGAGCAUCUC.

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